Neuroprotective peptides and uses thereof

ABSTRACT

The invention relates to neuroprotective peptides which bind calcium and which are useful in treating stroke and other neurodegenerative diseases, as well as compositions containing such peptides. The peptides preferably are conjugated to or administered with a compound which facilitates delivery across the blood-brain barrier.

This application is a continuation of and claims priority to applicationSer. No. 09/810,863, filed Mar. 16, 2001, entitled “NeuroprotectivePeptides and Uses Thereof,” now allowed, which is a divisional of andclaims priority to application Ser. No. 09/021,247, filed Feb. 10, 1998,entitled “Neuroprotective Peptides and Uses Thereof,” now U.S. Pat. No.6,225,444, issued May 1, 2001, the entire disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to isolated peptides which are useful in treatingstroke and other neurodegenerative diseases. The isolated peptides alsoare useful for binding calcium. The peptides preferably are conjugatedto a compound which facilitates delivery across the blood-brain barrier.

BACKGROUND OF THE INVENTION

Approximately 750,000 new strokes occur in the United States every yearand cause about 250,000 deaths (Kittner et al., J. Am. Med. Assoc.264:1267-1271, 1990). While the human suffering caused by stroke isenormous, both to the victims and their families, the economic costs areenormous as well. Long-term follow-up studies show that most strokesurvivors experience permanent disability ranging from loss ofvocational competence (71%), to requiring assistance with daily care(31%), to institutionalization (16%) (Gresham et al., N. Eng. J Med.293:954-959, 1975). Based on these data, roughly 300,000 personspermanently lose some function each year because of stroke.

The fundamental hypothesis in stroke research is that ischemia producesdisability and death, not directly, but rather indirectly by initiatinga cascade of cellular processes that eventually lead to neuronal death(Pulsinelli et al., Annals Neurol. 1 1:499-509, 1981; Choi, TrendsNeurosci. 11:465-469, 1988). Until physicians can regenerate functionalneurons to replace dead ones, the best hope for stroke victims is tointervene quickly with treatments that interrupt and reverse the cascadeof events triggered by the primary ischemic event before they becomeirreversible.

The cascade of events begins about three to four minutes after ischemia:the first step is that the concentration of extracellular excitatoryamino acids increases by 10- to 100-fold (Mayevsky, Brain Res. 524:1-9,1990; Mitani and Katoaka, Neuroscience 42:661-670, 1991). Theseexcitotoxic amino acids trigger a subsequent chain of events thatincludes calcium release from intracellular stores and eventually theexpression of new genes. Dead neurons and irreversible loss of cognitiveand behavioral function are results of this cascade which occurs hoursafter the initial ischemia.

A goal of anti-stroke treatment is to intervene in the cascade ofneuronal death before it becomes irreversible, saving as many neurons aspossible. A substantial body of work indicates that this theoreticalpossibility is a realistic goal. For example, several naturallyoccurring proteins can prevent neuronal death after excitotoxic damagein vitro or after experimental ischemia in vivo (Berlove et al., Soc.Neurosci. 17:1267, 1991; Shigeno et al., J. Neurosci. 11:2914-2919,1991). These proteins (including nerve growth factor, brain derivedneurotrophic factor, basic fibroblast growth factor, ciliaryneurotrophic factor, and others) derive from two structurally relatedprotein families, neurotrophins and cytokines, and are involved in thecontrol of neuronal differentiation in the central and peripheralnervous system. The most likely mechanism by which these proteinsprotect neurons from ischemia seems to involve the expression of variousgenes. Presumably those gene products inhibit a cell death program whichis triggered by the excitotoxins, and which could involve calciumrelease from intracellular stores. One of the most interesting previousfindings shows that some of these neurotrophic factors can protectneurons from death when applied up to tens of minutes after the injury(Shigeno et al., 1991).

Other examples of compounds used to treat the neurodegenerative effectsof cerebral ischemia include U.S. Pat. No. 5,559,095, which describes amethod of treating ischemia-related neuronal damage usingomega-conotoxin peptides and related peptides which bind to and blockvoltage-gated calcium channels, and U.S. Pat. No. 4,684,624, whichdescribes treatment using certain opioid peptides. These peptides arenot related to neurotrophins or cytokines.

While the neuroprotective effects of the neurotrophins are encouraging,their potential clinical application is limited by their large size (10kD or greater) which prevents effective delivery through the blood-brainbarrier (BBB). Neuroprotective molecules that can cross the BBB to acton neurons imperiled by cerebral ischemia will be more efficacious inthe treatment of stroke. Molecules that protect neurons against theischemic effects of stroke will also be useful for treating Alzheimer'sdisease, as well as the memory deficits that are characteristic of theaging process.

SUMMARY OF THE INVENTION

It has now been discovered that peptides can be derived from aneurotrophin and maintain the neuroprotective capabilities of the largerprotein. Peptides that maintain the neuroprotective effects ofependymin, a protein from which amino acid sequence of the peptides ispartially derived, have been prepared. It has also been discovered thatpeptides which conform to the EF-hand rule of calcium binding proteinsare neuroprotective.

According to one aspect of the invention, a composition comprising anisolated peptide is provided. The peptide includes the amino acidsequence set forth in SEQ ID NO:1. In certain embodiments, the isolatedpeptide includes the amino acid sequence of SEQ ID NO:2. In otherembodiments, the isolated peptide binds calcium. In still otherembodiments, the isolated peptide lacks one or more calcium coordinationresidues of the amino acid sequence of SEQ ID NO:1. Preferably, theforegoing isolated peptides include the amino acid sequence set forth inSEQ ID NO:3, and more preferably consists essentially of the amino acidsequence set forth in SEQ ID NO:3.

According to another aspect of the invention, a composition comprisingan isolated peptide is provided. The isolated peptide includes the aminoacid sequence set forth in SEQ ID NO:19 and in certain embodimentsincludes the amino acid sequence set forth in SEQ ID NO:10. In preferredembodiments, the isolated peptide includes the amino acid sequence setforth in any of SEQ ID Nos:11-18. Preferably the foregoing isolatedpeptides bind calcium.

In the foregoing compositions, the isolated peptide also can include 1-6amino acids on one or more of the N-terminus and the C-terminus of theisolated peptide, wherein the amino acids are selected from the groupconsisting of lysine and arginine. In certain of the embodiments ofthese compositions, the isolated peptide comprises 2-4 lysines and/orarginines on the N-terminus or the C-terminus of the isolated peptide.In preferred embodiments, the isolated peptide is selected from thegroup consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:9, mostpreferably SEQ ID NO:4.

In the foregoing compositions, the isolated peptide also can includefatty acids. Preferred fatty acids include docosahexaenoic acid.

In certain embodiments of the foregoing compositions, the isolatedpeptide is non-hydrolyzable, which means that the peptide bonds are lessreadily hydrolyzed than peptide bonds formed between L-amino acids.Preferred non-hydrolyzable peptides include those selected from thegroup consisting of peptides comprising D-amino acids, peptidescomprising a -psi[CH₂NH]— reduced amide peptide bond, peptidescomprising a -psi[COCH₂]— ketomethylene peptide bond, peptidescomprising a -psi[CH(CN)NH]— (cyanomethylene)amino peptide bond,peptides comprising a -psi[CH₂CH(OH)]— hydroxyethylene peptide bond,peptides comprising a -psi[CH₂O]— peptide bond, and peptides comprisinga -psi[CH₂S]— thiomethylene peptide bond. The most preferred isolatedpeptides are those which include 1-3 D-amino acids.

In the foregoing compositions, the isolated peptide is between 4 and 25amino acids in length and preferably is between 10 and 20 amino acids inlength.

In some embodiments of the invention, the isolated peptide is conjugatedto a compound which facilitates transport across the blood-brain barrierinto the brain. A blood brain barrier transport compound preferably isselected from the group consisting of docosohexaenoic acid, atransferrin receptor binding antibody, cationized albumin,Met-enkephalin, lipoidal forms of dihydropyridine, and cationizedantibodies.

According to another aspect of the invention, a method for treating asubject having a condition characterized by cerebral ischemia isprovided. The method includes administering to the subject an amount ofan isolated peptide which includes the amino acid sequence of SEQ IDNO:1 effective to reduce the neurotoxic effect of cerebral ischemia inthe subject. In certain embodiments, the isolated peptide isadministered to the subject after the cerebral ischemia event. In otherembodiments, the isolated peptide includes an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:5. The isolated peptide also can be conjugated to acompound which facilitates transport across the blood-brain barrier intothe brain, or the method can include administering a compound whichincreases transport across the blood-brain barrier.

In another aspect of the invention, a method for increasing neuronalcell AP-1 or NF-IL6 transcription factor activity in a subject isprovided. The method includes administering to the subject an amount ofan isolated peptide which includes the amino acid sequence of SEQ IDNO:1 effective to increase the activity of AP-1 or NF-IL6 in thesubject. In some embodiments, the isolated peptide includes an aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4 and SEQ ID NO:5. The isolated peptide also can beconjugated to a compound which facilitates transport across theblood-brain barrier into the brain, or the method can includeadministering a compound which increases transport across theblood-brain barrier.

According to still another aspect of the invention, a pharmaceuticalcomposition is provided. The pharmaceutical composition includes anisolated peptide which comprises the amino acid sequence set forth inSEQ ID NO:1, and a pharmaceutically acceptable carrier. Preferably, thepeptide reduces the neurotoxic effect of cerebral ischemia. Thepharmaceutical composition also can include a compound which facilitatestransport across the blood-brain barrier into the brain, which compoundcan be conjugated to the isolated peptide.

According to another aspect of the invention, a method for bindingcalcium is provided. The method includes contacting a calcium containingenvironment with one of the foregoing compositions, preferably acompostion which includes an isolated peptide which includes the aminoacid sequence set forth in SEQ ID NO:10.

Another aspect of the invention provides a method for identifying acalcium-binding peptide. The method includes providing a putativecalcium-binding peptide, contacting the putative calcium-binding peptidewith an environment containing calcium, and determining the calciumbinding of the peptide. In certain embodiments, the putative calciumbinding peptide is a variant of the amino acid sequence set forth in SEQID NO:1 or SEQ ID NO:19. In other embodiments, the step of providing aputative calcium-binding peptide includes providing a library havingpeptides including the amino acid sequences set forth in SEQ ID NO:1and/or SEQ ID NO:19.

Another aspect of the invention provides a method for identifying apeptide which increases AP-1 or NF-IL6 transcription factor activity.The method includes the steps of providing a peptide, contacting thepeptide with a cell which can express AP-1 or NF-IL6 transcriptionfactor activity, and determining the AP-1 or NF-IL6 transcription factoractivity to identify peptides which increase AP-1 or NF-IL6transcription factor activity. In certain embodiments, the peptide is avariant of the amino acid sequence set forth in SEQ ID NO:1 or SEQ IDNO:19. In other embodiments, the step of providing a peptide includesproviding a library having peptides including the amino acid sequencesset forth in SEQ ID NO:1 and/or SEQ ID NO:19.

According to another aspect of the invention, an isolated nucleic acidis provided. The nucleic acid encodes one of the foregoing isolatedpeptides. Also included in the invention are vectors, such as expressionvectors, which include the isolated foregoing isolated nucleic acids.

The use of the foregoing compositions, isolated peptides and isolatednucleic acids in the preparation of medicament also in provided.

These and other aspects of the invention are described in greater detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the eight day records of the general locomotor activity(GLA) of gerbils after forebrain ischemia.

FIG. 2 shows the effect of NMI 9236 on the GLA of gerbils afterforebrain ischemia.

FIG. 3 shows the effect of intravenous injections of NMI 9236 at 1 hrpost-ischemia on the GLA of gerbils.

FIG. 4 shows a comparison of the survival of different populations ofhippocampal neurons in gerbil brains after forebrain ischemia.

FIG. 5 contains photographs showing cross-sections of gerbil brainsillustrating the effects of ischemia on CA1 hippocampal neuron survival.

FIG. 6 shows the effect of NMI 9236 on the stimulation of AP-1 andNF-IL6 transcription factors in neuroblastoma cultures.

FIG. 7 shows competition with unlabeled AP-1 probe for AP-1 induced byNMI 9236.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions comprising isolated peptides. Theisolated peptides are characterized in that they can reduce theneurodegenerative effects of a cerebrovascular ischemic event (e.g.,stroke) when administered before or after the ischemic event. Thus,administration of the compositions of the invention reduces the loss ofthe neurons which follows a cerebrovascular ischemic event. Inparticular, as demonstrated below, administration of isolated peptidesreduces the loss of CA1 neurons of the hippocampus. The isolatedpeptides also can bind calcium efficiently.

As used herein, “isolated” means a peptide described herein is not in anatural state (e.g. it is disassociated from a larger protein moleculein which it naturally occurs), or is a non-naturally occurring fragmentof a naturally occurring protein (e.g. the peptide comprises less than25%, preferably less than 10% and most preferably less than 5% of thenaturally occurring protein). Isolated also may mean that the amino acidsequence of the peptide does not occur in nature, for example, becausethe sequence is modified from a naturally occurring sequence (e.g. byalteration of calcium binding amino acids), or because the sequence doesnot contain flanking amino acids which are present in nature.

An isolated peptide can be purified from a biological extract, preparedin vitro by recombinant or synthetic means, and/or modified byattachment of a moiety (e.g. a fluorescent, radioactive, or enzymaticlabel, or an unrelated sequence of amino acids to make a fusion protein)which does not correspond to a portion of the peptide in its nativestate. Isolated peptides include chimeric proteins comprising a fusionof an isolated peptide with another peptide, e.g., a peptide capable oftargeting the isolated peptide to a cell type or tissue type, enhancingstability of the isolated peptide under assay conditions, or providing adetectable moiety, such as green fluorescent protein. A moiety fused toan isolated peptide or a fragment thereof also may provide means ofreadily detecting the fusion protein, e.g., by immunological recognitionor by fluorescent labeling. Purified isolated peptides include peptidesisolated by methods including, but are not limited to,immunochromotography, HPLC, size-exclusion chromatography, ion-exchangechromatography and immune-affinity chromatography.

Likewise, “isolated” as used in connection with nucleic acids whichencode peptides embraces all of the foregoing, e.g. the isolated nucleicacids are disassociated from adjacent nucleotides with which they areassociated in nature, and can be produced recombinantly, synthetically,by purification from biological extracts, and the like. Isolated nucleicacids can contain a portion which encodes a one of the foregoingpeptides and another portion which codes for another peptide or protein.The isolated nucleic acids also can be labeled. Preferably the nucleicacids include codons which are preferred for mammalian usage. In certainembodiments, the isolated nucleic acid is a vector, such as anexpression vector, which includes a nucleic acid which encodes one ofthe foregoing isolated peptides.

In certain embodiments the isolated peptides have an amino acid sequenceincluding SEQ ID NO:1. Using single letter amino acid abbreviations, thepeptide is represented as: SEQ ID NO:1 D - X - D - X - D - X - A - X -D - X - X - E Q       N       S       D       E           QH       T       G       F       G           AY       E       N       K       S           L                L       T       T           N                        Y       M                         R       N                        V                         C                        SEach vertical column represents amino acids which can be substituted ateach position. Each X indicates that any amino acid can be used in theposition. Substitution at “X” positions with amino acids which do notdecrease the neuroprotective effects of the neuroprotective peptides arepreferred; several examples are given below.

In certain embodiments, the isolated peptide is a calcium-bindingpeptide, the sequence of which fits the EF-hand rule (see, e.g., Tuftyand Kretsinger, Science 187:167-169, 1975). For example, in SEQ ID NO:1,it is believed that the six amino acid residues which are restricted inamino acid composition (positions 1, 3, 5, 7, 9 and 12) form anoctahedral structure (“cage”) that in its three-dimensional conformationchelates calcium ions. In calcium binding embodiments of the isolatedpeptides, the amino acids at positions 2, 4, 6, 8, 10 and 11 can be anyamino acid which does not alter the secondary or tertiary structure ofthe peptide in a way that calcium ion binding is significantly reducedor eliminated.

For example, calcium binding peptide sequences based on the EF-hand ruleand SEQ ID NO:1 include the following sequence: D-X-D-X-D-G-X-I-D-X-X-E(SEQ ID NO:2). This peptide can have any amino acid at the “X”positions, although preferred amino acids are those which do notsubstantially reduce the calcium ion binding by the peptide.

In certain instances it can be advantageous to reduce the calciumbinding of the isolated peptides. Peptides having a reduced bindingaffinity for calcium ions can be prepared by making changes to theEF-hand octahedral cage. This can be accomplished generally by varyingthe amino acid sequence of the neuroprotective peptide at positionswhich form the octahedral cage. For example, isolated peptides whichvary from SEQ ID NO:1 or SEQ ID NO:2 by deletion of one or more of theterminal calcium coordination residues can be prepared. One simplyprepares a peptide which lacks one, two, three or four N-terminal orC-terminal residues involved in EF-hand calcium coordination. This typeof substitution results in a peptide which has a reduced length ascompared to the “parent” peptide, and which forms a partial octahedralcage. Preferably no more than two calcium binding residues are altered,more preferably no more than one calcium binding residue is altered, andmost preferably no calcium binding residue is altered.

Thus in some embodiments the isolated peptides comprise the amino acidsof SEQ ID NO:19, and in certain preferred embodiments comprise the aminoacids of SEQ ID NO:10. For example, the peptide comprising the aminoacid sequence set forth in SEQ ID NO:10 has been shown to chelatecalcium as tightly as the peptide of SEQ ID NO:3. In addition, suchpeptides can have amino acids added at either end of SEQ ID NO:10.Preferably amino acids are added in accordance with SEQ ID NO:1 and SEQID NO:3. For example, when one amino acid is added to SEQ ID NO:10, itpreferably is added to the N-terminus, and can be any amino acid (e.g.the “X” at position 4 of SEQ ID NO:1). More preferably, the X is aglycine, in accordance with position 4 of SEQ ID NO:3. When anotheramino acid is added to SEQ ID NO:10 to make a 10 amino acid peptide, itpreferably is added to the N-terminus, and preferably is a D, N, T or Eresidue. More preferably, the amino acid is a D, in accordance withposition 3 of SEQ ID NO:3. When a third amino acid is to SEQ ID NO:10 tomake an 11 amino acid peptide, it preferably is added to the N-terminus,and can be any amino acid (e.g. the “X” at position 2 of SEQ ID NO:1).More preferably, the X is a glycine, in accordance with position 2 ofSEQ ID NO:3. When a fourth amino acid is added to SEQ ID NO:10 to make a12 amino acid peptide, it preferably is added to the N-terminus, andpreferably is a D, Q, G or Y residue. More preferably, the amino acid isa D, in accordance with position 1 of SEQ ID NO:3.

Calcium binding by the isolated peptides also can be reduced byreplacing an internal calcium binding amino acid of the EF-handoctahedral cage (i.e., non-terminal cage amino acid) with a non-calciumbinding amino acid. For example, referring to the sequence of SEQ IDNO:1, one could substitute at the fifth position an amino acid which isnot a D, S, G, N or L. This type of substitution results in a peptidewhich has the same length as the “parent” peptide, but which forms anoctahedral cage missing one coordination site. Similar substitutions canbe made at more than one coordination site.

A particularly preferred peptide is D-G-D-G-D-F-A-I-D-A-P-E (SEQ IDNO:3), which generally fits the EF-hand rule, except that the seventhposition is not an aspartic acid residue, and thus is an example of the“internal” substitution of the EF-hand octahedral cage described above.This peptide exhibits neuroprotective activity as demonstrated in theExamples below. The design of the C-terminal portion of this peptide wasbased on a loose similarity to a portion of the neuronal growth factorependymin. The peptide itself acts as a growth stimulatory molecule,inducing the expression of transcription factors which bind to specificpromoter sequences in the genome. It is believed that thesetranscription factors, AP-1 and NF-IL6, are active in regulation of cellgrowth and apoptosis mechanisms, the balance of which can affect thegrowth of neuronal cells.

Peptides which include both “terminal” and “internal” substitutions inthe EF-hand octahedral cage also can be prepared. An example of apeptide combining “terminal” and “internal” modifications is the peptideD-F-A-I-D-A-P-E (SEQ ID NO:10). This peptide chelates calcium eventhough it is lacking the two N-terminal coordination sites of thepeptide set forth in SEQ ID NO:3.

Any of the foregoing peptides can be tested for calcium binding by wellknown assays of calcium chelation (see, e.g. Cornell-Bell et al.,Science 247:470-473, 1990; Cornell-Bell et al., Cell Calcium 12:185-204,1991). For example, one preferred method employs the calcium sensitivedye fura-2 to measure the chelation of calcium by the isolated peptides.In such an assay, cells are loaded with fura-2 and calcium. In thepresence of calcium ions, fura-2 exhibits a characteristic emissionspectrum when exposed to excitation radiation of appropriatewavelengths. An isolated peptide then is added to the cells and thediminution of fura-2 fluorescence is determined. Dose responseexperiments can be performed to determine the concentration at which theisolated peptide completely eliminates fura-2 fluorescence. For example,the isolated peptide of SEQ ID NO:4 completely eliminates fura-2fluorescence at a concentration of I pg/ml in the cell culture medium.The peptide of SEQ ID NO:10 which lacks three of the EF-hand calciumcoordination sites is about half as effective as the peptide of SEQ IDNO:4.

One preferred peptide (SEQ ID NO:3) was originally designed based on thesequence of the neurotrophic protein ependymin. Other neurotrophinproteins also can be used as the basis for preparation of isolatedpeptides which can be neuroprotective and/or calcium binding peptides.The neurotrophin-derived peptides can be assessed for neurotrophinactivity in tests which specifically measure the neuroprotectiveactivity of a particular neurotrophin (e.g., promoting survival ofneurons in culture, etc). Thus, it will be recognized by those ofordinary skill in the art, that other peptides will exist that functionas described and can be easily isolated according to the methods of theinvention.

Other preferred isolated peptides vary from the foregoing sequences bythe addition of basic amino acids at one or both ends of the peptide. Ingeneral, one to six lysine or arginine residues, or mixtures thereof,can be added to any of the foregoing peptides at either the N-terminus,the C-terminus, or both termini. Preferably, two to four lysines and/orarginines are incorporated at one or both ends of the isolated peptide.Exemplary peptides include SEQ ID NO:4 (K-K-D-G-D-G-D-F-A-I-D-A-P-E),SEQ ID NO:5 (K-K-K-K-D-G-D-G-D-F-A-I-D-A-P-E) and SEQ ID NO:9(K-K-K-K-D-G-D-G-D-F-A-I-D-A-P-E-K-K-K-K).

The amino acid sequence of isolated peptides may be of natural ornon-natural origin, that is, they may comprise a natural peptidemolecule that is a piece of a naturally occurring molecule, may comprisea sequence modified from a naturally occurring molecule, or may beentirely synthetic as long as the peptide has the ability to protectneurons from degradation following a cerebrovascular ischemic event,increases AP-1 or NF-IL6 transcription factor activity, and/or retainsthe property of binding calcium ions. Isolated peptides of the inventionalso may be altered versions of the foregoing. For example, isolatedpeptides in this context may be fusion proteins of a neuroprotectivepeptide and unrelated amino acid sequences, synthetic peptides of aminoacid sequences shown in SEQ ID NOs:1-5, 9, 10 and 19, labeled peptides,peptides coupled to nonpeptide molecules (for example in certain drugdelivery systems) and other molecules which include the amino acidsequences of SEQ ID Nos: 1-5, 9, 10 and 19.

The isolated peptides can be prepared as libraries having sequences setforth in SEQ ID NO:1 or SEQ ID NO:19. For example, a library ofsemi-random octapeptides based on SEQ ID NO:19 can be prepared asfollows. Conventiently, the peptides can be covalently attached to beads(e.g., polystyrene), with or without a linker (such as Gly-Gly-Gly) sothat each bead contains a unique sequence. Attachment to beads canfacilitate isolation of individual peptides after screening the libraryfor peptides having a desired property.

-   -   Step One. The pool of beads is divided into 5 aliquots. The        first aliquot is reacted with Asp, the second with Ser, the        third with Gly, the fourth with Asn and the fifth with Leu.    -   Step Two. The five aliquots are combined and then divided into        twenty equal aliquots. Each of the aliquots is reacted with one        of the twenty amino acids.    -   Step Three. The twenty aliquots are combined and then divided        into 10 aliquots. Each of the aliquots is reacted with one of        the amino acids given for position three of SEQ ID NO:19.

Steps Four through Eight are performed in the same manner as the stepsabove to create the library of peptides corresponding to SEQ ID NO:19.The library then is screened for peptides having a particular property,such as calcium binding or induction of AP-1 activity. The properties ofthe peptides are screened according to standard procedures in the art,using the assays for function described herein. For example, the librarycan be divided into a number of aliquots, diluted to reduce the numberof peptides per sample, and samples tested for calcium binding. Sampleswhich bind calcium can be further divided and/or diluted until there areonly one or a few peptides per sample, and retested for calcium binding.The amino acid sequence of these peptides can be determined, and thepeptide(s) synthesized for testing clacium binding individually. Manyother methods for preparing and screening peptide libraries, includingphage display, are known to one of ordinary skill in the art and can beemployed to screen for the peptides described herein.

Phage display can be particularly effective in identifying isolatedpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a biased degenerate array asdescribed above, or may completely restrict the amino acids at one ormore positions (e.g., for a library based on SEQ ID NO:1). One then canselect phage-bearing inserts which bind calcium. This process can berepeated through several cycles of reselection of phage that bindcalcium. Repeated rounds lead to enrichment of phage bearing particularsequences. DNA sequence analysis can be conducted to identify thesequences of the expressed polypeptides. The minimal linear portion ofthe sequence that binds calcium can be determined. One can repeat theprocedure using a biased library containing inserts containing part orall of the minimal linear portion plus one or more additional degenerateresidues upstream or downstream thereof.

Preferably, the isolated peptides are non-hydrolyzable. As used herein,non-hydrolyzable means that the bonds linking the amino acids of thepeptide are less readily hydrolyzed than peptide bonds formed betweenL-amino acids. To provide such peptides, one may select isolatedpeptides from a library of non-hydrolyzable peptides, such as peptidescontaining one or more D-amino acids or peptides containing one or morenon-hydrolyzable peptide bonds linking amino acids. Alternatively, onecan select peptides which are optimal for a preferred function (e.g.neuroprotective effects, calcium binding) in assay systems described inthe Examples and then modify such peptides as necessary to reduce thepotential for hydrolysis by proteases. For example, to determine thesusceptibility to proteolytic cleavage, peptides may be labeled andincubated with cell extracts or purified proteases and then isolated todetermine which peptide bonds are susceptible to proteolysis, e.g., bysequencing peptides and proteolytic fragments. Alternatively,potentially susceptible peptide bonds can be identified by comparing theamino acid sequence of an isolated peptide with the known cleavage sitespecificity of a panel of proteases. Based on the results of suchassays, individual peptide bonds which are susceptible to proteolysiscan be replaced with non-hydrolyzable peptide bonds by in vitrosynthesis of the peptide. Preferably the non-hydrolyzable peptide bondsor amino acids do not alter the calcium binding and/or neuroprotectiveactivity of the peptides.

Many non-hydrolyzable peptide bonds are known in the art, along withprocedures for synthesis of peptides containing such bonds.Non-hydrolyzable bonds include -psi[CH₂NH]— reduced amide peptide bonds,-psi[COCH₂]— ketomethylene peptide bonds, -psi[CH(CN)NH]—(cyanomethylene)amino peptide bonds, -psi[CH₂CH(OH)]— hydroxyethylenepeptide bonds, -psi[CH₂O]— peptide bonds, and -psi[CH₂S]— thiomethylenepeptide bonds.

Nonpeptide analogs of peptides, e.g., those which provide a stabilizedstructure or lessened biodegradation, are also contemplated. Peptidemimetic analogs can be prepared based on a selected peptide byreplacement of one or more residues by nonpeptide moieties. Preferably,the nonpeptide moieties permit the peptide to retain its naturalconformation, or stabilize a preferred, e.g., bioactive, conformation.One example of methods for preparation of nonpeptide mimetic analogsfrom peptides is described in Nachman et al., Regul. Pept. 57:359-370(1995). Peptide as used herein embraces all of the foregoing.

Likewise, various changes may be made including the addition of variousside groups that do not affect the manner in which the peptidefunctions, or which favorably affect the manner in which the peptidefunctions. Such changes may involve adding or subtracting charge groups,substituting amino acids, adding lipophilic moieties that do not effectbinding but that affect the overall charge characteristics of themolecule facilitating delivery across the blood-brain barrier, etc. Foreach such change, no more than routine experimentation is required totest whether the molecule functions according to the invention. Onesimply makes the desired change or selects the desired peptide andapplies it in a fashion as described in detail in the examples. Forexample, if the peptide (modified or unmodified) is active in a test ofneurotrophin function, or if such a peptide competes with the parentneurotrophin in a test of neurotrophin function, then the peptide is afunctional neurotrophin peptide. If the peptide (modified or unmodified)is active in a test of calcium binding, then the peptide is a functionalcalcium binding peptide.

The invention also embraces functional variants of the isolated peptide.As used herein, a “functional variant” or “variant” of an isolatedpeptide is a peptide which contains one or more modifications to theprimary amino acid sequence of the isolated peptide and retains theproperties disclosed herein. Modifications which create a functionalvariant of the isolated peptide can be made, for example, 1) to enhancea property of an isolated peptide, such as peptide stability in anexpression system; 2) to provide a novel activity or property to anisolated peptide, such as addition of an antigenic epitope or additionof a detectable moiety; or 3) to provide a different amino acid sequencethat produces the same or similar peptide properties. Modifications toan isolated peptide can be made to a nucleic acid which encodes thepeptide, and can include deletions, point mutations, truncations, aminoacid substitutions and additions of amino acids. Alternatively,modifications can be made directly to the peptide, such as by cleavage,addition of a linker molecule, addition of a detectable moiety, such asbiotin, addition of a fatty acid, substitution of one amino acid foranother and the like. Modifications also embrace fusion proteinscomprising all or part of the isolated peptide amino acid sequence.

If a variant involves a change to an amino acid of SEQ ID Nos: 1-5, 9,10 or 19, then functional variants of the isolated peptide havingconservative amino acid substitutions typically will be preferred, i.e.,substitutions which retain a property of the original amino acid such ascharge, hydrophobicity, conformation, etc. Examples of conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

One of skill in the art will be familiar with methods for predicting theeffect on peptide conformation of a change in amino acid sequence, andcan thus “design” a variant isolated peptide which maintains a similarconformation according to known methods. One example of such a method isdescribed by Dahiyat and Mayo (Science 278:82-87, 1997), wherebyproteins can be designed de novo. The method can be applied to theisolated peptides described herein to vary only a portion of the aminoacid sequence. By applying the computational methods of Dahiyat andMayo, specific variants of isolated peptides can be designed,synthesized and then tested for function in the assays described hereinto determine whether the variant peptide retains a desired function.

Other methods for identifying functional variants of the isolatedpeptides are provided in a published PCT application of Strominger andWucherpfennig (US/96/03182). These methods rely upon the development ofamino acid sequence motifs to which potential epitopes may be compared.Each motif describes a finite set of amino acid sequences in which theresidues at each (relative) position may be (a) restricted to a singleresidue, (b) allowed to vary amongst a restricted set of residues, or(c) allowed to vary amongst all possible residues. For example, a motifmight specify that the residue at a first position may be any one of theresidues valine, leucine, isoleucine, methionine, or phenylalanine; thatthe residue at the second position must be histidine; that the residueat the third position may be any amino acid residue; that the residue atthe fourth position may be any one of the residues valine, leucine,isoleucine, methionine, phenylalanine, tyrosine or tryptophan; that theresidue at the fifth position must be lysine, and so on.

Sequence motifs for neuroprotective peptide functional variants can bedeveloped further by analysis of the peptide structure and conformationof the neuroprotective peptides disclosed herein. By providing adetailed structural analysis of the residues involved in forming thecontact surfaces of the neuroprotective peptides, one is enabled to makepredictions of sequence motifs which have similar binding properties.

Using these sequence motifs as search, evaluation, or design criteria,one is enabled to identify classes of peptides (functional variants ofthe isolated peptides disclosed herein) which have a reasonablelikelihood of binding to the target of the disclosed isolated peptidesand inducing a neuroprotective response and/or binding calcium. Thesepeptides can be synthesized and tested for activity as described herein.Use of these motifs, as opposed to pure sequence homology (whichexcludes many peptides which are functionally similar but quite distinctin sequence) or sequence homology with unlimited “conservative”substitutions (which admits many peptides which differ at criticalhighly conserved sites), represents a method by which one of ordinaryskill in the art can evaluate peptides for potential application in thetreatment of the neurodegenerative effects of cerebrovascular ischemia,stroke and the like.

Thus methods for identifying functional variants of an isolated peptideare provided. In general, the methods include selecting an isolatedpeptide, such as the isolated peptide comprising the amino acid sequenceof SEQ ID NO:3. A first amino acid residue of the isolated peptide ismutated to prepare a variant peptide. In one embodiment, the amino acidresidue can be mutated according to the principles set forth in theStrominger and Wucherpfennig PCT application described above. In otherembodiments, mutation of the first amino acid residue can be selectedand tested using computer models of peptide conformation. Peptidesbearing mutated residues which maintain a similar conformation (e.g.secondary structure) can be considered potential functional variantswhich can be tested for function using the assays described herein. Anymethod for preparing variant peptides can be employed, such as synthesisof the variant peptide, recombinantly producing the variant peptideusing a mutated nucleic acid molecule, and the like. The properties ofthe variant peptide in relation to the isolated peptides describedpreviously are then determined according to standard procedures asdescribed herein.

Variants of the isolated peptides prepared by any of the foregoingmethods can be sequenced, if necessary, to determine the amino acidsequence and thus deduce the nucleotide sequence which encodes suchvariants.

Isolated peptides such as those descibed above preferably are shortenough to be synthesized and isolated readily, yet long enough toeffectively reduce the neurodegenerative effects of cerebral ischemiaand/or bind calcium. Preferred peptides thus are between five andtwenty-five amino acids in length, e.g., 5, 6, 7, 8,9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.More preferably, peptides are between eight and twenty amino acids inlength. Those skilled in the art are well-versed in methods forpreparing and isolating such peptides, such as synthetic chemistry orrecombinant biological methods.

Peptides useful in the invention can be linear, or maybe circular orcyclized by natural or synthetic means. For example, disulfide bondsbetween cysteine residues may cyclize a peptide sequence. Bifunctionalreagents can be used to provide a linkage between two or more aminoacids of a peptide. Other methods for cyclization of peptides, such asthose described by Anwer et al. (Int. J Pep. Protein Res. 36:392-399,1990) and Rivera-Baeza et al. (Neuropeptides 30:327-333, 1996) are alsoknown to those of skill in the art.

Nonpeptide analogs of peptides, e.g., those which provide a stabilizedstructure or lessened biodegradation, are also contemplated. Peptidemimetic analogs can be prepared based on a selected peptide byreplacement of one or more residues by nonpeptide moieties. Preferably,the nonpeptide moieties permit the peptide to retain its naturalconformation, or stabilize a preferred, e.g., bioactive, conformation.One example of methods for preparation of nonpeptide mimetic analogsfrom peptides is described in Nachman et al., Regul. Pept. 57:359-370(1995). Peptide as used herein embraces all of the foregoing.

In some circumstances, it may be preferable to conjugate the isolatedpeptide to a compound which facilitates transport of the peptide acrossthe blood-brain barrier (BBB). As used herein, a compound whichfacilitates transport across the BBB is one which, when conjugated tothe peptide, facilitates the amount of peptide delivered to the brain ascompared with non-conjugated peptide. The compound can induce transportacross the BBB by any mechanism, including receptor-mediated transport,and diffusion.

Compounds which facilitate transport across the BBB include transferrinreceptor binding antibodies (U.S. Pat. No. 5,527,527); certain lipoidalforms of dihydropyridine (see, e.g., U.S. Pat. No. 5,525,727); carrierpeptides, such as cationized albumin or Met-enkephalin (and othersdisclosed in U.S. Pat. Nos. 5,442,043; 4,902,505; and 4,801,575);cationized antibodies (U.S. Pat. No. 5,004,697); fatty acids such asdocosahexaenoic acid (DHA; U.S. Pat. No. 4,933,324) and C8 to C24 fattyacids with 0 to 6 double bonds, glyceryl lipids, cholesterol,polyarginine (e.g., RR, RRR, RRRR) and polylysine (e.g., KK, KKK, KKKK).Unbranched, naturally occurring fatty acids embraced by the inventioninclude C8:0 (caprylic acid), C10:0 (capric acid), C12:0 (lauric acid),C14:0 (myristic acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid),C16:2, C18:0 (stearic acid), C18:1 (oleic acid), C18:1-7 (vaccenic),C18:2-6 (linoleic acid), C18:3-3 (α-linolenic acid), C18:3-5(eleostearic), C18:3-6 (&-linolenic acid), C18:4-3, C20:1 (gondoicacid), C20:2-6, C20:3-6 (dihomo-y-linolenic acid), C20:4-3, C20:4-6(arachidonic acid), C20:5-3 (eicosapentaenoic acid), C22:1 (docosenoicacid), C22:4-6 (docosatetraenoic acid), C22:5-6 (docosapentaenoic acid),C22:5-3 (docosapentaenoic ), C22:6-3 (docosahexaenoic acid) and C24:1-9(nervonic). Highly preferred unbranched, naturally occurring fatty acidsare those with between 14 and 22 carbon atoms. The most preferred fattyacid is docosahexaenoic acid. Other BBB carrier molecules and methodsfor conjugating such carriers to peptides will be known to one ofordinary skill in the art. Such BBB transport molecules can beconjugated to one or more ends of the peptide.

The isolated peptide can be conjugated to such compounds by well-knownmethods, including bifunctional linkers, formation of a fusionpolypeptide, and formation of biotin/streptavidin or biotin/avidincomplexes by attaching either biotin or streptavidin/avidin to thepeptide and the complementary molecule to the BBB-transport facilitatingcompound. Depending upon the nature of the reactive groups in anisolated peptide and a targeting agent or blood-brain barrier transportcompound, a conjugate can be formed by simultaneously or sequentiallyallowing the functional groups of the above-described components toreact with one another. For example, the transport-mediating compoundcan prepared with a sulthydryl group at, e.g., the carboxyl terminus,which then is coupled to a derivatizing agent to form a carriermolecule. Next, the carrier molecule is attached via its sulfhydrylgroup, to the peptide. Many other possible linkages are known to thoseof skill in the art.

Conjugates of a peptide and a targeting agent or BBBtransport-facilitating compound are formed by allowing the functionalgroups of the agent or compound and the peptide to form a linkage,preferably covalent, using coupling chemistries known to those ofordinary skill in the art. Numerous art-recognized methods for forming acovalent linkage can be used. See, e.g., March, J., Advanced OrganicChemistry, 4th Ed., New York, N.Y., Wiley and Sons, 1985), pp.326-1120.

For peptides which exhibit reduced activity in a conjugated form, thecovalent bond between the peptides and the BBB transport-mediatingcompound is selected to be sufficiently labile (e.g., to enzymaticcleavage by an enzyme present in the brain) so that it is cleavedfollowing transport of the peptides across the BBB, thereby releasingthe free peptides to the brain. Art-recognized biologically labilecovalent linkages, e.g., imino bonds, and “active” esters can be used toform prodrugs where the covalently coupled peptides is found to exhibitreduced activity in comparison to the activity of the peptides alone.Exemplary labile linkages are described in U.S. Pat. No. 5,108,921,issued to Low et al.

If a peptide does not have a free amino-or carboxyl-terminal functionalgroup that can participate in a coupling reaction, such a group can beintroduced, e.g., by introducing a cysteine (containing a reactive thiolgroup) into the peptide by synthesis or site directed mutagenesis.Disulfide linkages can be formed between thiol groups in, for example,the peptide and the BBB transport-mediating compound. Alternatively,covalent linkages can be formed using bifunctional crosslinking agents,such as bismaleimidohexane (which contains thiol-reactive maleimidegroups and which forms covalent bonds with free thiols). See also thePierce Co. Immunotechnology Catalogue and Handbook Vol. 1 for a list ofexemplary homo-and hetero-bifunctional crosslinking agents,thiol-containing amines and other molecules with reactive groups.

Other methods for covalently coupling the peptide to the derivatizingagent and/or to the extracellular agent include, for example, methodsinvolving glutaraldehyde (Riechlin, Meth. Enzymology 70:159-165, 1980);N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide (Goodfriend et al.,Science 144:1344-1346, 1964); and a mixture ofN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide and a succinylatedcarrier (Klapper and Klotz, Meth. Enzymol. 25:531-536, 1972). Ingeneral, the conjugated peptides of the invention can be prepared byusing well-known methods for forming amide, ester or imino bonds betweenacid, aldehyde, hydroxy, amino, or hydrazo groups on the respectiveconjugated peptide components. As would be apparent to one of ordinaryskill in the art, reactive functional groups that are present in theamino acid side chains of the peptide (and possibly in the BBBtransport-mediating compound) preferably are protected, to minimizeunwanted side reactions prior to coupling the peptide to thederivatizing agent and/or to the extracellular agent. As used herein,“protecting group” refers to a molecule which is bound to a functionalgroup and which may be selectively removed therefrom to expose thefunctional group in a reactive form. Preferably, the protecting groupsare reversibly attached to the functional groups and can be removedtherefrom using, for example, chemical or other cleavage methods. Thus,for example, the peptides of the invention can be synthesized usingcommercially available side-chain-blocked amino acids (e.g.,FMOC-derivatized amino acids from Advanced Chemtech Inc., Louisville,Ky.). Alternatively, the peptide side chains can be reacted withprotecting groups after peptide synthesis, but prior to the covalentcoupling reaction. In this manner, conjugated peptides of the inventioncan be prepared in which the amino acid side chains do not participateto any significant extent in the coupling reaction of the peptide to theBBB transport-mediating compound or cell-type-specific targeting agent.

Alternatively, it may be preferable to administer the peptides incombination with a compound which increases transport across theblood-brain barrier (BBB). Such compounds, which need not be conjugatedto a peptide, increase the transport of the peptide across the BBB intothe brain. A compound which increases transport across the BBB is one,for example, which increases the permeability of the BBB, preferablytransiently. Coadministration of a peptide with such a compound permitsthe peptide to cross a permeabilized BBB. Examples of such compoundsinclude bradykinin and agonist derivatives (U.S. Pat. No. 5,112,596);and receptor-mediated permeabilizers such as A-7 (U.S. Pat. Nos.5,268,164 and 5,506,206).

The isolated neuroprotective peptides described herein are characterizedby their ability to prevent the neurodegenerative effects of cerebralischemia. Although not wishing to be bound by any particular mechanism,it is believed that the peptides exert their neuroprotective effectsthrough one or both of the following mechanisms: regulation of theexpression of transcription factors such as AP-1 and NF-IL6 to reduceapoptosis of the neurons, and calcium ion binding to reduce theneurotoxic effects of calcium ions. These properties, as well asexperimental indicia of neuroprotection, provide a basis for making andtesting variant neuroprotective peptides. Indicia of neuroprotectioninclude (1) upregulation of AP-1 and/or NF-IL6, (2) calcium binding, (3)promotion of survival of neurons in culture and (4) protection of CA1hippocampal neurons following cerebral ischemia in a standard animalmodel of stroke.

Peptides, including variant peptides, can be tested for retention forany of the foregoing properties. For example, the peptides can be testedfor in vitro properties initially to determine which of the variantpeptides retain the ability to bind calcium ions and/or stimulate theexpression of transcription factors. In vitro assays of calcium bindinginclude contacting the peptide with an environment which containscalcium, such as a cell preloaded with calcium and a fluorescentcalcium-sensitive dye, and determining the calcium binding of thepeptide Peptides which retain one or more of these properties can thenbe used in in vivo assays of neuroprotection such as the Mongoliangerbil assay described below. Neuroprotective peptides or their variantswhich are conjugated to targeting compounds, labels, blood-brain barriercarriers and the like can be tested for retention of neuroprotectiveactivity as well as for the activity of the conjugated compound (e.g.,appropriate targeting, detectable labeling, ability to cross theblood-brain barrier, etc.).

For example, as exemplified below, the variant peptide can be used inassays which quantitate the expression of the transcription factors AP-1and NF-IL6. The variant peptides can also be tested for their ability topromote the growth and sprouting of neurons (described in Shashoua etal., J. Neurosci. Res. 32:239-244, 1992). Further, the variant peptidescan be tested for calcium ion binding according to standard assays suchas those employing calcium sensitive dyes. Finally, for variant peptidesthat exhibit characteristics similar to present neuroprotective peptidesin in vitro tests, in vivo tests of neuroprotection using the Mongoliangerbil model of stroke can be performed to evaluate the neuroprotectiveproperties of the variant peptides.

With respect to functional variant peptides, the methods also caninclude the step of comparing the neuroprotective properties of variantpeptides to the neuroprotective properties of one or moreneuroprotective peptides as a determination of the effectiveness of theneuroprotection by the functional variant peptide. By comparing thefunctional variant peptide with one or more neuroprotective peptides,variant peptides having enhanced neuroprotective properties can beselected.

Neuroprotective peptides are useful in the treatment of conditions whichare characterized by cerebral ischemia, such as stroke. Such peptidesalso are useful for the selection of other compounds which bind to anneuroprotective peptide binding molecule. For example, where theneuroprotective peptide is based on the amino acid sequence of aneurotrophin such as ependymin, the neuroprotective peptide can be usedin competition assays to select compounds which bind to ependyminbinding molecules more avidly than the peptide. The peptides are alsouseful in the design of other compounds for reducing theneurodegenerative effects of cerebral ischemia, such as small moleculeinhibitors, which are based on the molecular structure or conformationof the neuroprotective peptide. Thus, the peptides can be used in vivofor the treatment of disease, as well as in vitro for the design andtesting of compounds which reduce neurodegeneration and compounds whichbind neurotrophin molecules. The peptides can also be used to generateantibodies useful in diagnostic assays of neurotrophin expression.Finally, the peptides can be used to turn on transcription factors or tobind calcium.

Also a part of the invention are those nucleic acid sequences which codefor an isolated peptide or variant thereof and other nucleic acidsequences which hybridize to a nucleic acid molecule consisting of theabove described nucleotide sequences, under stringent conditions. Theterm “stringent conditions” as used herein refers to parameters withwhich the art is familiar. Nucleic acid hybridization parameters may befound in references which compile such methods, e.g. Molecular Cloning:A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers to hybridization at 65° C. inhybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% Polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 25 mM NaH₂PO₄ (pH7), 0.5% SDS,2 mM EDTA). SSC is 0.15M Sodium Chloride/0.15M Sodium Citrate, pH 7; SDSis Sodium Dodecyl Sulphate; and EDTA is Ethylene diaminetetraaceticacid. After hybridization, the membrane upon which the DNA istransferred is washed at 2×SSC at room temperature and then at0.1×SSC/0.1×SDS at 65° C.

There are other conditions, reagents, and so forth which can used, whichresult in a similar degree of stringency. The skilled artisan will befamiliar with such conditions, and thus they are not given here. It willbe understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of nucleic acids encoding the neuroprotectivepeptides of the invention. The skilled artisan also is familiar with themethodology for screening cells and libraries for expression of suchmolecules which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing. Vectors, includingexpression vectors, which include the foregoing nucleic acids also areincluded in the invention. One of ordinary skill in the art is familiarwith a variety of cloning and expression vectors, as well as methods forinserting a nucleic acid in a vector, and particularly for operablylinking a nucleic acid with a promoter sequence without introducing stopcodons, frame shifts or other mutations, to provide efficient expressionof the nucleic acid in an expression vector.

Compositions including isolated peptides, including the peptides havingsequences set forth in SEQ ID Nos:1-5, 9, 10 and 19, are administered toa subject to treat a condition characterized by neuronal degeneration.Such conditions include conditions characterized by cerebral ischemia,such as stroke, and other conditions characterized by progressiveneuronal degeneration, such as Alzheimer's disease. Isolated peptidesare administered to a subject in need of such treatment in an amounteffective to reduce the neuronal cell degeneration resulting from such acondition, e.g. stroke.

Peptides or other compounds which protect neurons following cerebralischemia may be administered as part of a pharmaceutical composition.Such a pharmaceutical composition may include the peptides incombination with any standard pharmaceutically acceptable carriers whichare known in the art. The compositions should be sterile and contain atherapeutically effective amount of the decoy peptides or othertherapeutic compound in a unit of weight or volume suitable foradministration to a patient. The term “pharmaceutically acceptable”means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Thecharacteristics of the carrier will depend on the route ofadministration. Pharmaceutically acceptable carriers include diluents,fillers, salts, buffers, stabilizers, solubilizers, and other materialswhich are well known in the art.

When used therapeutically, the compounds of the invention areadministered in therapeutically effective amounts. In general, atherapeutically effective amount means that amount necessary to delaythe onset of, inhibit the progression of, or halt altogether theparticular condition being treated. Therapeutically effective amountsspecifically will be those which desirably influence the survival ofneurons following stroke or other cerebral ischemic insult. Generally, atherapeutically effective amount will vary with the subject's age, andcondition, as well as the nature and extent of the disease in thesubject, all of which can be determined by one of ordinary skill in theart. The dosage may be adjusted by the individual physician,particularly in the event of any complication. A therapeuticallyeffective amount typically varies from 0.01 mg/kg to about 1000 mg/kg,preferably from about 0.1 mg/kg to about 200 mg/kg and most preferablyfrom about 0.2 mg/kg to about 20 mg/kg, in one or more doseadministrations daily, for one or more days.

The effect of the administered therapeutic composition can be monitoredby standard diagnostic procedures. For example, in the treatment of theneurodegeneration which follows a stroke, the administration of acomposition which includes neuroprotective peptides reduces thedegeneration of CA1 hippocampal neurons. The reduction of degenerationof CA1 hippocampal neurons following treatment can be assessed using MRIand CT scans. Where other indicia of neurodegeneration are available(such as the increase of locomotor activity demonstrated by theMongolian gerbil animal model of stroke), such indicia may also be usedin diagnosing neurodegeneration following treatment with the peptidecompositions.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, xylitol, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose or xylitol), and the like. Preservatives and other additivesmay also be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intracranial, intraperitoneal, intramuscular, intracavity,intrarespiratory, subcutaneous, or transdermal. The route ofadministration will depend on the composition of a particulartherapeutic preparation of the invention. Administration by intravenousinjection is preferred after the onset of a cerebral ischemic event suchas a stroke.

It is envisioned that the neuroprotective compositions described hereincan be delivered to neuronal cells by site-specific means.Cell-type-specific delivery can be provided by conjugating a peptide toa targeting molecule, e.g., one which selectively binds to the affectedneuronal cells. Methodologies for targeting include conjugates, such asthose described in U.S. Pat. No. 5,391,723 to Priest. Another example ofa well-known targeting vehicle is liposomes. Liposomes are commerciallyavailable from Gibco BRL (Gaithersburg, Md.). Numerous methods arepublished for making targeted liposomes. Liposome delivery can beprovided by encapsulating a decoy peptide in liposomes which include acell-type-specific targeting molecule. Methods for targeted delivery ofcompounds to particular cell types are well-known to those of skill inthe art.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the active compounds of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di andtriglycerides; hydrogel release systems; silastic systems; peptide basedsystems; wax coatings, compressed tablets using conventional binders andexcipients, partially fused implants and the like. In addition, apump-based hardware delivery system can be used, some of which areadapted for implantation.

A long-term sustained release implant also may be used. “Long-term”release, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well known to those of ordinary skill in the art andinclude some of the release systems described above. Such implants canbe particularly useful in treating conditions characterized by recurrentcerebral ischemia, thereby affecting localized, high-doses of thecompounds of the invention.

EXAMPLES

Experimental Methods:

(a) In vivo test methods: The gerbil (male Mongolian) has been widelyused as an experimental model for studies of ischemic stroke because thebrain blood supply is controlled by only two common carotid arteries.This unusual feature occurs in gerbils because they have an incompletecircle of Willis (Chandler et al., J. Pharmacol. Methods 14:137-146,1985; Finkelstein et al., Restor. Neurol.. Neurosci. 1:387-394, 1990;Levine and Sohn, Arch. Pathol. 87:315-317, 1969; Kahn, Neurology22:510-515, 1972). A five minute bilateral carotid occlusion results inneuronal cell death that is predominantly localized in the CA1 subfieldof the hippocampus. The CA1 neurons degenerate and virtually disappearby 7-8 days after ischemia (Akira, Res. in Immunol. 143:734-736 1992;Crain et al., Neurosci. 27:387-402, 1988). In addition, the gerbil modelhas the advantage that within one day after ischemia the animalsincrease by 100% to 200% their general locomotor activity. This readilymeasurable change can persist for at least eight days in most of theexperimental animals (Akira, 1992; Kuroiwa et al., Neurosci. Lett.122:141-144, 1991; Ohno et al., Eur. J. Pharmacol. 193:357-361, 1991;Phillis, Brain Res. Bull. 23:467-470, 1989). Such results indicate thatthe affected neurons are physiologically non-functional within one dayafter ischemia, even though significant histological changes may notappear in the brain until several days later.

(b) Surgicalprocedures. Male Mongolian gerbils were anesthetized with amixture of isoflurane and oxygen using an inhalation apparatus(Stoelting Instrument Co.). An incision was made in the ventral neck andthe common carotid arteries were isolated and occluded completely for aperiod of 5 minutes using microaneurysm clips. Next, the clips wereremoved and the incision sutured. The anesthesia was continued until theend of the i.v. injection and infusion period. Mean arterial bloodpressure was monitored by a polyethylene catheter implanted in the leftor right femoral vein during surgery. The core temperature of the gerbilwas controlled by a heating pad and heating lamp connected to a rectaltemperature probe (Model 73A, Yellow Springs Instruments). At the end ofthe infusion, the anesthesia was discontinued and the animal allowed torecover while the heating pad maintained body temperature.

(c) Behavioral assessment. Previous studies have demonstrated thatcerebral ischemia significantly elevates the spontaneous generallocomotor activity (GLA) in a gerbil (by about 2-fold or more) beginningwithin a few hours after the ischemia onset and continuing for at leasteight days (Gerhardt et al., Behav. Neurosci. 102:310-303, 1988; Kuroiwaet al., 1991; Phillis, 1989). This hyperactivity appears to correlatewith the extent of ischemic damage to the hippocampus and is reduced bypharmacological treatments (Kuroiwa et al., 1991; Phillis 1989). Thus,GLA analyses can be used as a relatively quick behavioral indicator ofthe efficacy of the potency of a pharmacological treatment in rescuinghippocampal neurons from ischemic damage. GLA analyses, in conjunctionwith detailed histological data, were used to evaluate the efficacy ofNMI 9236 for treatment of ischemic stroke.

GLA measurements were carried out in half hour sessions using theStoelting Electronic Activity monitor (Shashoua et al., J. Med. Chem.27:654-659, 1984; Jacob et al., J. Med. Chem. 30:1573-1576, 1987). Eachgerbil's activity level was first assessed on days 3 and 1 prior to theexperimental ischemia procedure to obtain a base line GLA data, then ondays 1, 2, 5, and 7 after the carotid artery occlusion to determineefficacy of drug treatment. All test sessions were conducted at the sametime of day. On day 8, the animals were sacrificed and their brainsremoved for histopathological analysis.

(d) Histological procedures: At the time of the sacrifice, each animalwas deeply anesthetized and perfused transcardially with heparinizedsaline followed by paraformaldehyde (4%) in phosphate buffered saline.After fixation the brains were placed in 30% sucrose for 3 days,embedded in glutaraldehyde-gelatin, cut frozen into 30μ serial sectionsfor morphometric analysis, and stained with cresyl violet. The celldensities per 1000 microns2 were determined by computer assistedcounting of grey level images at 300× viewed through an Axioplanmicroscope. It was determined that a maximum of 62% of the hippocampalpyramidal cells survived the ischemia in the presence of NMI 9236 ascompared to 4% survival for the control saline non-drug treated animals.All analyses were carried out blind.

Example 1 Synthesis of Peptide NMI 9236

The 14 amino acid peptide (SEQ ID NO:4; NMI 9236), with side chainprotection in place, was first synthesized by the Merrifield process (J.Am. Chem. Soc. 86:304, 1963). N-substituted docosohexaenoic acid (DHA)derivatives of NMI 9236 were synthesized by reacting DHA anhydride withthe N-terminal residue of the peptide in the presence of4-dimethylaminopyridine. Briefly, peptide NMI 9236 (bound to resin) waswashed twice with 20 ml DMF and then 20 ml of DMF containing 20%piperidine (Aldrich Chemical Co.) was added. The mixture was stirred byan argon gas stream for 10 minutes. The product was filtered and washedthrice with DMF and thrice with methylene chloride. The treatedresin-bound peptide was combined with 30 ml CH₂Cl₂, 20 ml DHA anhydridein benzene and 0.15 g 4-dimethylaminopyridine. The mixture was stirredwith argon gas for 5 hours. The product was filtered, washed 4 timeswith 30 ml CH₂Cl₂, dried and stored at 4° C. overnight. To release anddeprotect the modified peptide, the resin was mixed with 20 ml 95/5TFA/phenol and 2 ml mercaptoethanol; the mixture was allowed to stand atroom temperature for 24 hours. Released peptide was purified by HPLC.About 10 mg peptide was synthesized and used in tests in the gerbilmodel for ischemic stroke.

Example 2 Evaluation of the Properties of NMI 9236

In Vivo Protection of Ischemic Stroke by Intracranial Administration:

In the first experiments, the peptides were delivered directly into thebrain via an intracranial cannula to establish bioactivity using thegerbil model for ischemic stroke.

Male Mongolian gerbils were anesthetized with a mixture of halothane andoxygen in Phase 1 of surgery and a cannula (an Alzet No. 2002 pump) wasimplanted subcutaneously in the midscapular region beneath the skin ofthe animal. Such pumps can deliver 0.5 microliters per hour through thecannula for a period of up to 2 weeks. The output of this pump wasinserted into the left lateral ventricle through a bore hole secured tothe skull with acrylic cement. All surgical procedures were carried outwith a strict control of the temperature of each animal; rectaltemperatures were monitored and heat was supplied via a temperatureregulated heating pad. After recovery from surgery during a period of 4days, the animals were again anesthetized and an incision was made intothe ventral neck and the common carotid arteries were isolated andoccluded completely for a period of 5 minutes using microaneurysmclamps. These were then removed and the incision was sutured to completethe surgery.

The animals were then studied in three groups: Group 1 received salinefrom the Alzet pump via intracerebroventricular (icv) delivery. Theserepresented a control in which the maximum damage from the ischemia didoccur. Group 2 received the peptide as a solution of 1 milligram per mlin normal saline. Group 3 was a sham control experimental group in whichthe surgery was identical to the other two groups, but the carotidarteries were not occluded and no ischemia took place. Each Alzet pumpcontained a volume of 0.3 ml for delivery during a 14-day period, 7 dayspre- and 7 days post-ischemia.

At day 1 through day 8 post-ischemia, the spontaneous general locomotoractivity (GLA) of the gerbils was measured for 1 hour in an activitymonitor apparatus (Stewart et al., 1978). It has been established inprevious studies of the gerbil model that the spontaneous locomotoractivity is elevated by two or more fold as a result of ischemic damage.At 8 days after ischemia, the CA1 hippocampal neurons die and disappearfrom the brain. Bilateral damage occurs from this ischemia and an animalbecomes hyperactive (see FIG. 1). This behavior is detectable at day 1post surgery as a result of damage to both left and right CA1hippocampal neuronal subfields. If one side of the brain is intact, noincrease in GLA is obtained (Kuroiwa et al., 1991; Phillis et al.,1989). Thus, the spontaneous GLA measurements can be used as a rapidbehavioral indicator of the development of ischemia and for anassessment of the efficacy of a pharmacological treatment for rescue ofneurons from neurotoxic effects.

FIGS. 1 and 2 show a summary of the data for animals from each testgroup (n=4). The GLA for each animal in a group is compared to its ownGLA measured at one day prior to the surgery (day 1 data was used as astandard), and day 0 was the surgical day. It was observed thatintercerebroventricular (icv) delivery of saline to ischemic gerbils atday 1 post surgery doubled the GLA to an average value of 200%. The GLAdecreased to 80% for the group that received NMI 9236, indicating thatthe peptide had a neuroprotective effect. This GLA result was identical,within experimental error, to the GLA data obtained for the shamoperated controls.

These results were confirmed by histological analysis (see FIGS. 4 and5). At 8 days post surgery the gerbils were sacrificed, perfusion fixedwith formalin, and each perfused brain was embedded inglutaraldehyde-gelatin. About 300 serial sections (40 microns thick)were cut from each frozen brain, and stained with cresyl violet. Cellmorphometric analysis was carried out on one out of every 10 sections bycomputer-assisted cell density counting of grey level images viewed at300× through an Axioplan microscope. Focused camera input (Sony CCD)from the microscope to the IBS video screen of the Zeiss IBAS/KONTRONImage analysis system was normalized and segmented before assessing cellnumber. The average cell density present per a.1000 micron² region ofthe CA1 and CA3 pyramidal cell layers for the left and right side ofeach were determined. The analysis was carried out blind.

FIG. 4 presents a summary of the results. The data for the left CA1 andright CA1 sides of the NMI 9236 treated brains showed a somewhatunexpected but highly useful result (see FIG. 4 and 5). Only the CA1cells located on the left side which received the direct output of theNMI 9236 peptide from the Alzet pump were rescued from the ischemia(52±12% survival of L-CA1 neurons); CA1 cells on the right side weredead and eliminated from the brain during the 8 day post-ischemia period(4±2% survival of R-CA1 neurons). These results suggest that the peptideeither was destroyed by proteases before it reached the right CA1 regionor that insufficient amounts of the peptide arrived at the right side toproduce neuroprotection. One consequence of this observation was thateach brain section served as its own control, thereby removing anydoubts about whether an ischemia was actually generated in a givenbrain. This result is shown in FIG. 4, where the cell density found forthe ischemic brains (saline controls) was only 0.6 for both left (L-CA1)and right (R-CA1) and comparable to the right CA1 level of the NMI 9236treated brain, i.e., the unprotected side. The L-CA1 of the peptidetreated brain had a high cell density count of 6.2, i.e., about 50% ofthe level found for the sham operated control brain. Also, the fact thatthe cell densities of the left and right CA3 were identical in eachbrain section represented an additional internal control for thehistological processing. FIG. 5 shows schematic diagrams of thehistology of a representative section from one brain from each of thethree groups studied, illustrating the recovery of the L-CA1 but not theR-CA1 cells (FIG. 5C) in the peptide NMI 9236 treated brain and avirtually complete loss of CA1 neurons in the non-peptide (saline)treated brain (FIG. 5B).

The data for treatment with NMI 9236 also demonstrated adequate drugdelivery to only one side of the brain. This distribution controls forthe occasional false positive data that are due to the presence of athird blood supply to the brains of some animals. Such animals wouldhave both the left and right CA1 fields remaining intact. No examples ofgerbils with anomalous brain blood supply have been found in theseexperiments. The foregoing results suggest that the peptide NMI 9236 wasneuroprotective when administered in vivo.

Example 3 Intravenous Delivery of Peptide NMI 9236

For a drug to be useful for a stroke patient, the drug preferablyprovides neuroprotection when administered after an ischemic stroke,since one generally cannot know when such an event can occur. In aninitial experiment of delivery of NMI 9236 by intravenous (i.v.)injections, it was demonstrated that the peptide blocked the developmentof enhanced GLA if it was administered at 10 minutes after the ischemicevent (n=3, see FIG. 1). In a second series of experiments, the effectsof delivery of peptide at a dose of 1 mg/kg at 1 hour after theocclusion of the forebrain cerebral arteries were investigated. FIG. 3summarizes these results. The gerbils in the peptide treated, controlnon-drug saline treated, and sham operated groups were injected with a50 microliter aliquot of the appropriate solution into the femoral veinat 1 hour after the surgery. The average GLA (85% of the day 1 data)value for the NMI 9236 treated group was identical to that for the shamoperated controls (see FIG. 3). The average GLA value for the salinetreated control group was 225%, a result consistent with severedestruction of hippocampal neurons. FIG. 3 also shows another control inwhich the peptide was given at 1 hour after the surgery as asubcutaneous (s.c.) injection. The GLA value obtained for the group was200%, indicating that there was no demonstrable neuroprotectionefficacy, as assayed by spontaneous general locomotor activity, when thepeptide was delivered via a s.c. route. Presumably, the peptide wasdestroyed by proteases before it could enter into the bloodstream andbegin to gain access to the brain. The non-hydrolyzable peptide analogsdescribed elsewhere herein are not susceptible to such degradation andthus can be delivered s.c. as well as by other modes of delivery.

These results suggest that the peptide NMI 9236 protected neurons froman ischemic insult when delivered to the brain by an intravenous route,even when delivered at 1 hour after the onset of ischemia.Neuropathological confirmation of this finding is summarized in Table 1.The results of cell counts indicate the delivery of NMI 9236 as an i.v.bolus was highly effective in rescuing the CA1 hippocampal neurons fromcell death. The analysis of serial sections of the drug treated brainsthat were subjected to the global ischemia showed that essentially allthe cells remained intact when the drug was delivered at 1 hour posttrauma. The control ischemic brains that received a bolus of the vehiclehad over 90% cell loss in the CA1 region of hippocampus. These findingssuggested that the preferred route of drug administration is intravenousinjection although other routes and modes of delivery, describedelsewhere herein, also are acceptable. TABLE 1 Analysis of Cell Densityof Gerbil Brains Subjected to Global Ischemia--i.v. Drug Delivery DataCell Density--number of cells/1000 microns² CA1-HippocampalCA3-Hippocampal Brain Type n field field Controls 3 12.1 + 1.1  8.4 +1.1 (sham operated) Ischemic (a) 3 0.8 + 0.7 11.1 + 1.2  (i.v. vehicle)Ischemic (b) 3 13.3 + 0.82 8.82 + 0.42 (1 mg/kg NMI 9236 i.v. @ 1 hrpost ischemia)All animals were sacrificed on day 8 following the ischemia or the shamoperation. The cell density data are the averages for 20 sections (oneevery 10th from serial sections of the hippocampus brain region).Gerbils were subjected to a 5 min. bilateral carotid artery occlusion togenerate ischemic stroke conditions.(a) The animals received a 50 μl i.v. bolus of the vehicle(physiological saline) at 1 hr post ischemia. This resulted in of 90%neuronal cell destruction in CA1 with no loss in CA3 regions of thehippocampus.(b) The animals received a 50 μl i.v. bolus of NMI 9236 at dose of 1mg/kg in physiological saline at 1 hr post ischemia. No significantneuronal losses were detectable in either the CA1 or the CA3 regions ofthe hippocampus in comparison to the sham operated controls.

Example 4 Studies of the Molecular Mechanism of Action of Peptide NMI9236

In previous work it was demonstrated that NMI 9236 promoted the growthand sprouting of neurons to at least the same extent as its 68kilodalton parent protein (Shashoua et al., 1992). In the presentstudies, neuroblastoma cultures were used to investigate the molecularmechanism of action of peptide NMI 9236.

It was determined that NMI 9236 can turn on specific genes related toneuronal growth in neuroblastoma tissue culture experiments. NB2a mouseneuroblastoma cultures treated with 5-50 μg/ml of NMI 9236 showed anincrease in the level of two transcription factors, AP-1 and NF-IL6, inthe nuclei of the cells, whereas NF-KB was not activated.

Western blots were used to assay the activation of various proteinkinases. Using electrophoretic mobility shift assays (EMSAs), timecourse and dose response experiments were performed to identifytransient activation events.

Based on the type of kinases and transcription factors effected by NMI9236, predictions were made concerning the main signal transductionpathways switched on by the drug, and the types of gene products likelyto be activated. Such gene products were then screened by Northern blothybridization using probes unique to those specific mRNAs to monitor thesteady state levels of specific mRNAs activated by NMI 9236 (Adams etal., J. Mol. Biol. 187:465-478, 1986; Adams et al., Gene 54:93-103,1987). Several types of control experiments were carried out toestablish that the stimulation by NMI 9236 is due to the peptide itself.The effect of the fatty acid carrier (DHA) was studied as one control.

The activation of specific transcription factors was assayed using anelectrophoretic mobility shift assay (EMSA, for review see: Kerr, Meth.Enzymol. 254:619-632, 1995). Nuclear extracts were prepared fromstimulated neuroblastoma cell cultures (10⁷ cells/sample) usingclassical nuclear extraction protocols (Dignam et al., Nucl. Acids Res.11: 1475-1489, 1983; Prywes and Roeder, Cell 47: 777-784, 1986). A 0.5pmol aliquot of a synthetic ³²P labeled oligomer duplex (with a sequenceknown to bind a specific transcription factor, see Table 2) was mixedwith 3 μg of nuclear extract protein, and the mixture incubated at roomtemperature for 20 minutes. Subsequent electrophoresis undernon-denaturing conditions through 4% polyacrylamide gels were used toseparate and resolve the high MW protein/DNA complexes (transcriptionfactor/DNA oligomer duplex) form low MW uncomplexed DNA oligomers.Autoradiography was used to visualize and quantify the complexes formed(see FIG. 6). TABLE 2 Transcription factor Oligonucleotide Sequences SEQID NO NF-κB 5′ AGTTGAGGGGACYFFCCAGGC 6 NF-IL6 5′ TGCAGATTGCGCAATCTGCA 7AP-1 (c-jun) 5′ CGCTFFGATGAGTCAGCCGGAA 8

Using the electrophoretic mobility shift assay (EMSA) we tested forchanges in concentration of transcription factors AP-1 and NF-IL6 inneuroblastoma nuclei as a function of stimulation by peptide NMI-9236.These two factors are well known to function in cell proliferation anddifferentiation, and to be activated by protein kinase-C. FIG. 6 showsthe EMSA data. Neuroblastoma cells, exogenously treated with peptide NMI9236 for 20 hours, showed a strong activation of AP-1 relative tocontrol (middle lanes in upper left panel). The activation is lesspronounced at 30 min (left lanes). Cultures incubated with the peptidein serum-free medium for 20 hours to remove serum growth factors showeda very strong activation of AP-1 (right lanes). Identification of thetwo observed bands as Jun/Fos heterodimers and Jun/Jun homodimers wasbased on their electrophoretic migration, as well as antibody studies(data not shown). Densitometric scans of the EMSA (see bottom left)showed the AP-1 activation to be at least 3-5 fold, which is significantsince 1.5 fold activation of AP-1 has been demonstrated to switch ontranscription. NF-κB, in similar experiments, was not stimulated,indicating that there was a specificity to the action of NMI-9236.

Demonstration that the observed EMSA bands were AP-1 family members wasconfirmed by a cold probe competition experiment (FIG. 7). The AP-1signal was partially competed by as little as 3-9 fold excess cold AP-1probe, and totally competed by 18-37 fold excess (left lanes), while a37 fold excess (right lanes) mutant AP-1 oligo only partly competed.

FIG. 6 also shows the EMSA data for NF-IL6 (upper right panel).Neuroblastoma cells exogenously treated with peptide NMI 9236 for 20hours showed a strong activation of NF-IL6 relative to control (middlelanes). The activation was less pronounced at 30 min (left panel).Cultures incubated with the peptide in serum-free medium for 20 hours toremove serum growth factors also showed a very strong activation ofNF-IL6 (right lanes). Identification of the observed band as NF-IL6(C/EBPa) was based on its electrophoretic migration, as well as antibodystudies (data not shown). Densitometric scan of the EMSA (FIG. 6, lowerright lanes) showed the NF-IL6 activation was at least 3-5 fold byincubation with 1 μg/ml of peptide NMI 9236 in comparison to control.

Example 5 Effect of Neuroprotective Peptides on Glutamate InducedNeurotoxicity In Vitro

The effect of the neuroprotective peptides was tested on rat brainhippocampal cells. Rat brain hippocampi were isolated by dissection of18 day old rat fetuses. Cells were isolated as described (Mattson andKater, J. Neurosci. 7:4034-4043, 1987; Mattson et al., J. Neurosci.8:2087-2100,1988; Mattson and Kater, Int. J. Dev. Neurosci. 6:439-452,1988). Peptide NMI 9236 was added in the culture medium at the indicatedconcentration when the cells were plated (in 10 cm culture dishes). Thecultures were then incubated for 30 min at 37° C. after which glutamatewas added at the concentrations indicated. Cells were counted after 3days of culture; healthy cells and total cells were counted. Table 3reports the results of the experiments, which results demonstrate thatNMI 9236 reduced the neurotoxicity induced by glutamate. TABLE 3Additions to hippocampal neuron cultures. Plate Treatment Total cellsHealthy cells % healthy 1 none (control) 105 101 96 2 1 mM glutamate 6835 51.5 3 2 mM glutamate 156 62 40.8 4 2 mM glutamate + 98 80 81.6 18μg/ml peptide 5 2 mM glutamate + 150 138 92 12 μg/ml peptideEquivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references cited herein are incorporated by reference in theirentirety.

A Sequence Listing is presented below and is followed by what isclaimed.

1 A method for increasing neuronal cell AP-1 or NF-IL6 transcriptionfactor activity in a subject, comprising administering to the subject anamount of an isolated peptide which comprises the amino acid sequence ofSEQ ID NO:1 effective to increase the activity of AP-1 or NF-IL6 in thesubject. 2 The method of claim 1, wherein the isolated peptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. 3 The method of claim 2,wherein the isolated peptide is conjugated to a compound whichfacilitates transport across the blood-brain barrier into the brain. 4 Amethod for binding calcium comprising contacting a calcium containingenvironment with the composition of claim
 3. 5. The method of claim 4,wherein the isolated peptide comprises the amino acid sequence set forthin SEQ IDNO:10. 6 A method for identifying a calcium-binding peptidecomprising providing a putative calcium-binding peptide, contacting theputative calcium-binding peptide with an environment containing calcium,and determining the calcium binding of the peptide. 7 The method ofclaim 6, wherein the putative calcium binding peptide is a variant ofthe amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:19.
 8. Themethod of claim 6, wherein the step of providing a putativecalcium-binding peptide comprises providing a library comprisingpeptides having the amino acid sequence set forth in SEQ ID NO:1 or SEQID NO:19. 9 A method for identifying a peptide which increases AP-1 orNF-IL6 transcription factor activity, comprising providing a peptide,contacting the peptide with a cell which can express AP-1 or NF-IL6transcription factor activity, and determining the AP-1 or NF-IL6transcription factor activity to identify the peptide which increasesAP-1 or NF-IL6 transcription factor activity.
 10. The method of claim 9,wherein the peptide is a variant of the amino acid sequence set forth inSEQ ID NO:1 or SEQ ID NO:19. 11 The method of claim 9, wherein the stepof providing a peptide comprises providing a library comprising peptideshaving the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:19.